Biosurfactants are amphiphilic molecules produced by several microorganisms including bacteria, filamentous fungi and yeasts. They are classified according to their chemical composition (glycolipids, lipopeptides, glycoproteins, glycolipopeptides, phospholipids) and their molecular weight (low and high molecular weight). Biosurfactants are currently used in several industrial fields (food industry, pharmacy, medicine, detergents, agriculture, cosmetics, oil recovery and bioremediation) owning to some of their specific properties such as their low toxicity, biocompatibility, biodegradability, environmentally-friendly, stability under extreme conditions (temperature, pH and salinity), structural diversity, production using renewable low-cost substrates, agreement with green chemistry and sustainability. Despite the properties of biosurfactants, there are few studies leading to their structural characterization. The most reported structures are from biosurfactants produced by Bacillus spp. (surfactins, fengycins, lichenysines) and Pseudomonas spp. (rhamnolipids). The majority of researches performed on biosurfactants emphasized their functional groups characterization. These biosurfactants have yet to be fully characterized at structural level. Hence, in this review, we highlight the different purification and structural characterization techniques which can be combined to provide information on the structure of the biosurfactants independently of its complexity. This will lead to enhance their application in some highly pointed industrial fields such as medicine and pharmacy.